The development of effective treatments, therapies or diagnostics for Apicomplexan parasite diseases has been hampered by lack of suitable models for study. Apicomplexans are parasitic protozoa that modulate host immune systems to favor infection of the host. It is known that Trypanosomes bind to muscarinic cholinergic receptors of host cells and attenuate secretion of immune regulator effector molecules. Identification of receptors and their ligands used by parasitic protozoa are important for understanding disease.
Apicomplexan parasites such as Sarcorystis neurona also cause disease by manipulating immune effector molecules, cytokines, that facilitate infections. Illustratively, Equine Protozoal Myeloencephalitis (EPM) which is the leading infectious neurologic equine disease in the Western Hemisphere is caused by the Apicomplexan parasite Sarcorystis neurona (S. neurona). The signs of disease caused by S. neurona are related to neuroinflammation. In most cases the parasite is no longer evident in the hosts central nervous system during chronic disease. Cytokine mediated inflammation is the predominantly recognized lesion. There is an intricate relationship between parasite invasion of leukocytes and cytokine responses to infection that dramatically impact expression of parasite genes. Parasite induced host cell cytokines drive proinflammatory responses and these responses may regulate genes that regulate parasite stage conversion in the host.
While the symptoms and effects of EPM have been recognized since the 1970's, it was not until 1991 that the protozoan parasite that causes EPM was isolated and cultured from a horse and given the name Sarcorystis neurona. Sarcorystis neurona cycles naturally between opossums and armadillos/raccoons. The feces of the opossum (the definitive host) is likely the source of the infection for horses. Thus, the horse is an aberrant host, becoming exposed when it consumes infectious material from opossum feces. In the horse, the most prominent EPM-producing organism, S. neurona, does not produce clinical signs of disease as a result of cyst formation, but as the cysts (sporozoites) convert to merozoites which in turn stimulate leukocytes that release cytokines in response to the infection. These cytokines can cross the blood brain barrier where they are proinflammatory. Clinical signs in a horse with EPM do not develop until the merozoite interacts with a functional white blood cell stimulating release of molecules that are responsible for inflammation.
The importance of leukocytes in the pathogenesis of sarcocystosis is evident by experiment. Severe combined immunodeficient (SCID) foals supported prolonged visceral infections and a parasitemia (from ingestion of S. neurona sporocysts) but did not develop clinical signs of EPM. Merozoite challenge (IV) using S. neurona also produced prolonged visceral infections without clinical signs of EPM in SCIO foals. In these SCIO foals (the absence of functional leukocytes) parasites did not enter the CNS despite the persistence of infections. Infection challenge of immunocompetent foals with S. neurona sporocysts result in a rapidly controlled parasitemia followed by clinical signs but no parasites enter the CNS. From the foregoing, it is evident that the interplay between the leukocyte and parasite are necessary to allow parasite stage conversion (to a stage that can enter the CNS via a leukocyte) and production of innate immune responses responsible for neuroinflammation.
Serum cytokines are the products of immune reaction to parasitic protozoa. Cytokines can cross the blood brain barrier within the central nervous system where they are proinflammatory. In an immunocompetent horse, the initial merozoite stage resulting in a parasitemia is quickly controlled. Some merozoites enter leukocytes and are transported to the CNS and can cause lesions in these tissues. Often parasites are not found in the CNS but neuroinflammation is recognized on histopathological examination.
Neuroinflammation is recognized in. the clinically ill animal. These signs include weakness, muscle atrophy, spinal ataxia, or “wobbling” and/or head tilt with asymmetry of the face (e.g., eyelid, ear, or lip). A severely EPM-affected horse may go down and be unable to rise. Lameness not traceable to orthopedic disease or any combination of the above signs may occur in early or less severe infections. In most cases, an affected horse is bright and alert with a normal appetite, hematological and biochemical blood values are usually in the normal range.
Epidemiology and economic significance of S. neurona infection is substantial. Of animals clinically affected, 30-40% reportedly fail to respond to current therapy, and some of these animals die. Conventional therapy relies on drug/medications and/or combinations that target organelles specific to protozoa. The efficacy of treatments cannot be optimized because of the lack of a model of the disease that can define molecular targets. Better and more effective prophylactic, or therapeutic modalities are required but were not thoroughly investigated before an animal model predictably produced disease. Unexpectedly, binding cholinergic receptors using levamisole HCl in the autologous leukocyte model of infection induced a parasitemia with no clinical signs as observed in the SCID infection experiment. Such a method can be used to identify molecular targets that are the progenitors of disease induced by S. neurona and will be applicable to other apicomplexans that cause neuroinflammation.
There are other animal induced infection models for S. neurona. An infection of nude mice or interferon gamma knock-out mice with sporocysts or culture derived merozoites produced infections. These experiments result in neurological signs and isolation of the organism from the CNS. However, the relevancy of this model is doubtful since these mice are immune-deficient; thus any immune-based selection forces acting in normal animals are absent. Similarly the model that used SCID foals that supported prolonged visceral infections and a parasitemia (from ingestion of S. neurona sporocysts) but did not develop clinical signs of EPM suffer because immune based selection forces acting in normal animals are absent. One animal model, which has been used to date to study S. neurona isolated from natural cases of EPM, uses autologous leukocytes that have been previously infected in vitro with S. neurona. This Trojan Horse model produces clinical signs and allows parasites to enter the CNS of horses (as was shown by culture).
From the foregoing, it would be realized that despite a great deal of past and on-going effort, there remains an unfulfilled need for animal models for Apicomplexan parasitic diseases that induce neuroinflammation.